The co-translational addition of N-linked oligosaccharide chains is critical for the proper folding, intracellular translocation and function of many membrane glycoproteins mediating essential neurobiological functions in the central nervous system (CNS). Because of the importance of this modification, the long-term goal of this project has been to understand the role of ER proteins in the regulation of biosynthesis of dolichyl phosphate (Dol-P), the assembly of Glc3Man9GlcNAc2-P-P-dolichol and the transbilayer movement of dolichyl-phospho-saccharide intermediates in brain. This application proposes five Specific Aims using biochemical and genetic approaches designed to: 1) isolate and learn more about the structures of ER proteins mediating the transverse diffusion of Man-P-Dol, Glc-P-Dol and ManSGlcNAc2 P-P-Dol in brain using a novel and promising transport assay with water-soluble analogues; 2) use biochemical and genetic approaches in a topological study of the assembly of a membrane-anchored mannan in M. luteus as a model system for membrane proteins involved in the synthesis and transbilayer movement of mannolipid intermediates in the ER in brain; 3) clone cDNAs that encode the long-chain cis-isoprenyltransferase (cis- IPTase) and dolichol kinase, two enzymes involved in the de novo biosynthesis of Dol-P in the CNS. These cloned cDNAs will provide coding sequence information and probes to be used to assess developmental changes in the expression of cis-IPTase and dolichol kinase in embryonic rat brain cultures and LPS-activated murine B cells; 4) isolate a temperature-sensitive mutant for dolichol kinase to test the hypothesis that this enzyme catalyzes the terminal step in the de novo pathway for Dol-P biosynthesis and 5) to extend the study of a new development in isoprenoid biosynthesis by investigating the enzymatic mechanism by which farnesol (F-OH) and geranylgeraniol (GG-OH) are converted to the "activated" isoprenyl donors, farnesyl pyrophosphate (F-P-P) and geranylgeranyl pyrophosphate (GG-P-P) , respectively in brain.